The isoflavonoids of the Leguminosae are among the most important biologically active classes of phenylpropanoid-derived plant natural products. Isoflavones such as daidzein, genistein and biochanin A exhibit a wide range of pharmacological effects including estrogenic, antiangiogenic, antioxidant and anticancer activities (Dixon, R. A. 1999. “Isoflavonoids: biochemistry, molecular biology and biological functions.” In U. Sankawa, eds, Comprehensive Natural Products Chemistry, Elsevier, pp 773-823), and the health promoting activity of high soy diets are believed to reside in their isoflavone components (Barnes, et al. 1990. “Soybeans inhibit mammary tumors in models of breast cancer.” In M. W. Pariza, eds, Mutagens and Carcinogens in the Diet, Wiley-Liss, Inc, New York, pp 239-253; Adlercreutz, et al. 1991. “Urinary excretion of lignans and isoflavonoid phytoestrogens in Japanese men and women consuming a traditional Japanese diet,” Am J Clin Nutr 54: 1093-1100; Lee, et al. 1991. “Dietary effects on breast-cancer risk in Singapore,” Lancet 337: 1197-1200). Daidzein and genistein act as precursors in the biosynthesis of various antimicrobial isoflavonoid phytoalexins in a wide variety of legumes (Dixon, R. A. and N. L. Paiva. 1995. “Stress-induced phenylpropanoid metabolism,” Plant Cell 7: 1085-1097). Furthermore, due to their estrogenic activity, high levels of isoflavonoids such as the phytoestrogen formononetin can adversely affect the reproductive capacity of sheep grazing forage legumes (Shutt, D. A. 1976. “The effects of plant oestrogens on animal reproduction,” Endeavour 75: 110-113).
Isoflavonoids have been ascribed key roles in plant-pathogen interactions because many have quite strong antimicrobial activity. Antimicrobial isoflavonoids fall into two functional classes, the pre-formed “phytoanticipins” and the inducible “phytoalexins” (VanEtten, et al. 1994. “Two classes of plant antibiotics: phytoalexins versus phytoanticipins,” The Plant Cell 6: 1191-1192). Examples of the former class include the prenylated isoflavones of lupin, which are synthesized in various organs of the plant during seedling development (Ingham, et al. 1983. “Fungitoxic isoflavones from Lupinus albus and other Lupinus species,” Zeitschrift für Naturforschung, C 38: 194-200). Examples of the latter include several pterocarpans, the biosynthesis of which has been studied in detail, particularly in the cases of phaseollin, medicarpin, pisatin and glyceollin from bean, alfalfa, pea and soybean, respectively (Dixon, R. A. 1999. “Isoflavonoids: biochemistry, molecular biology and biological functions,” Comprehensive Natural Products Chemistry, Vol. 1, U. Sankawa, ed, Elsevier, pp. 773-823).
Isoflavonoid compounds have been shown to accumulate in infected plant cells to levels known to be antimicrobial in vitro. The temporal, spatial and quantitative aspects of accumulation are consistent with a role for these compounds in disease resistance (Rahe, J. E. 1973. “Occurrence and levels of the phytoalexin phaseollin in relation to delimitation at sites of infection of Phaseolus vulgaris by Colletotrichum lindemuthianum” Can J Botany 51: 2423-2430; Hadwiger, L. A. and D. M. Webster. 1984. “Phytoalexin production in five cultivars of pea differentially resistant to three races of Pseudomonas syringae pv. Pisi,” Phytopathology 74: 1312-1314; Long, et. al. 1985. “Further studies on the relationship between glyceollin accumulation and the resistance of soybean leaves to Pseudomonas syringae pv. Glycinea,” Phytopathology 75: 235-239; Bhattacharyya, M. K. and E. W. Ward. 1987. “Biosynthesis and metabolism of glyceollin I in soybean hypocotyls following wounding or inoculation with Phytophthora megasperma f. sp. Glycinea,” Physiol Mol Plant Pathol 31: 387-405). Inhibition of the synthesis of glyceollin by application of an inhibitor of L-phenylalanine ammonia-lyase (PAL) to soybean seedlings breaks resistance to Phytophthora megasperma f. sp. glycinea (Moesta, P. and H. Grisebach. 1982. “L-2-Aminooxy-3-phenylpropionic acid inhibits phytoalexin accumulation in soybean with concomitant loss of resistance against Phytophthora megasperma f. sp. Glycinea,” Physiol Plant Pathol 21: 65-70). Isolates of the fungal pathogen Nectria hematococca with reduced ability to degrade the pea phytoalexin pisatin have reduced virulence on pea, suggesting that pisatin is functionally involved in the disease resistance response (Kistler, H. C. and H. D. VanEtten. 1984. “Regulation of pisatin demethylation in Nectria haematococca and its influence on pisatin tolerance and virulence,” J Gen Micro 130: 2605-2613).
Phytoalexins accumulate more rapidly, and to higher levels, during resistant interactions between a plant and its microbial pathogens than during susceptible interactions that result in disease (Dixon, R. A., and M. J. Harrison. 1990. “Activation, structure and organization of genes involved in microbial defense in plants,” Adv Genet 28: 165-234). Therefore, increasing the rate at which phytoalexins accumulate, and the absolute levels attained, would result in increased resistance (Lamb, et al. 1992. “Emerging strategies for enhancing crop resistance to microbial pathogens,” Bio/technology 10: 1436-1445).
Certain methylated forms of isoflavones, for example formononetin (7-hydroxy-4′-methoxyisoflavone) and biochanin A (5,7-dihydroxy-4′-methoxyisoflavone) have been shown to provide a nutraceutical benefit. For example, biochanin A and formononetin are reported to be phytoestrogens, and biochanin A has been shown to be effective in animal cancer study models. (Yangihara, et al. 1993. “Antiproliferative effects of isoflavones on human cancer cell lines established from the gastrointestinal tract,” Cancer Res 53:581 5-5821; and Zhou-Jin-Rong, et al. 1998. “Inhibition of murine bladder tumorigenesis by soy isoflavones via alterations in the cell cycle, apoptosis, and angiogenesis,” Cancer Res 58: 5231-5238).
As seen from the above examples, genetic manipulation of isoflavonoid biosynthesis in transgenic plants would positively impact plant, animal and human health (Dixon, et al. 1999. “Molecular controls for isoflavonoid biosynthesis in relation to plant and human health,” Recent Advances in Phytochemistry 33: 133-160). However, the enzymes responsible for increasing isoflavonoid phytoalexin accumulation have not been identified, and not all the genes encoding the enzymes of isoflavonoid phytoalexin biosynthesis have been cloned.
The biosynthetic branch pathway leading to isoflavones in plants involves a cytochrome P450 mediated 2-hydroxylation/aryl migration of a flavanone intermediate formed from phenylpropanoid- and acetate-derived precursors via the chalcone synthase and chalcone isomerase reactions (FIG. 1) (Kochs, G. and H. Grisebach. 1986. “Enzymic synthesis of isoflavones,” Eur J Biochem 155: 311-318; Hakamatsuka, et al. 1991. “P-450-dependent oxidative rearrangement in isoflavone biosynthesis: reconstitution of P-450 and NADPH:P450 reductase”, Tetrahedron 47: 5969-5978; Steele, et al. 1999. “Molecular characterization of the enzyme catalyzing the aryl migration reaction of isoflavonoid biosynthesis in soybean,” Arch Biochem Biophys 367: 146-150; and Jung, et al. 2000. “Identification and expression of isoflavone synthase: the key enzyme for biosynthesis of isoflavones in legumes,” Nature Biotechnology 18:208-212). After aryl migration, the 2-hydroxy isoflavanone intermediate undergoes dehydration to yield the corresponding isoflavone (Hakamatsuka, et al. 1998. “Purification of 2-hydroxyisoflavanone dehydratase from the cell cultures of Pueraria lobata,” Phytochemistry 49: 497-505). Genistein (4′,5,7-trihydroxyisoflavone) is the product of aryl migration/dehydration of naringenin (4′,5,7-trihydroxyflavanone), whereas daidzein (4′,7-dihydroxyisoflavone) is formed in a similar manner from liquiritigenin (4′,7-dihydroxyflavanone). 4′-O-Methylation of daidzein yields formononetin (7-hydroxy-4′-methoxyisoflavone), whereas 4′-O-methylation of genistein yields biochanin A (5,7-dihydroxy-4′-methoxyisoflavone), an important anticancer compound found in chickpea (Yanagihara, et al. 1993. “Antiproliferative effects of isoflavones on human cancer cell lines established from the gastrointestinal tract,” Cancer Res 53: 5815-5821) (FIG. 1).
In alfalfa and certain other legumes such as chickpea, methylation of the 4′-hydroxyl is a prerequisite for further substitutions of the isoflavonoid nucleus leading to pterocarpan phytoalexins such as medicarpin (Dixon, R. A. 1999. “Isoflavonoids: biochemistry, molecular biology and biological functions,” In U. Sankawa, eds, Comprehensive Natural Products Chemistry, Elsevier, pp 773-823). This reaction is important biotechnologically, because it represents a pathway entry point for conversion of isoflavones with human anticancer activity into downstream metabolites with antifungal activity for the plant. However, the exact mechanism of this O-methylation reaction has remained unclear. Based on radiolabeling studies in copper-induced alfalfa seedlings in which formononetin, but surprisingly not daidzein, was incorporated into the pterocarpan medicarpin, it has been proposed that the 4′-O-methylation might be an integral part of the aryl migration reaction of isoflavone biosynthesis (Dewick, P. M. and M. Martin. 1979. “Biosynthesis of pterocarpan, isoflavan and coumestan metabolites of Medicago sativa: chalcone, isoflavone and isoflavanone precursors,” Phytochemistry 18: 597-602). However, aryl migration occurs in vitro in the absence of methylation (Kochs, G. and H. Grisebach. 1986. Eur J Biochem 155: 311-318; Hakamatsuka, et al. 1991. Tetrahedron 47: 5969-5978; Kessmann, et al. 1990. “Stress responses in alfalfa (Medicago sativa L.) III. Induction of medicarpin and cytochrome P450 enzyme activities in elicitor-treated cell suspension cultures and protoplasts,” Plant Cell Rep 9: 3841; Steele, et al. 1999. “Molecular characterization of the enzyme catalyzing the aryl migration reaction of isoflavonoid biosynthesis in soybean,” Arch Biochem Biophys 367: 146-150), and mutants of subterranean clover exist in which formononetin and biochanin A are virtually absent, and daidzein and genistein accumulate instead (Wong, E. and C. M. Francis. 1968. “Flavonoids in genotypes of Trifolium subterraneum-II. Mutants of the Geraldton variety,” Phytochemistry 7: 2131-2137), indicating that the isoflavones are the natural substrates for 4′-O-methylation. The problem has also been compounded by the fact that it has not proven possible to purify a S-adenosyl-L-methionine (SAM) dependent O-methyltransferase (OMT) that can catalyze 4′-O-methylation of daidzein. Instead, the SAM-dependent isoflavone OMT from alfalfa (He, X-Z and R. A. Dixon. 1996. “Affinity chromatography, substrate/product specificity and amino acid sequence analysis of an isoflavone O-methyltransferase from alfalfa (Medicago sativa L.),” Arch Biochem Biophys 336: 121-129) produces 7-O-methyldaidzein (isoformononetin) in vitro by methylation of the hydroxyl group at the 7-position. Isoformononetin is a rarely occurring plant natural product that has not been reported from alfalfa.
The isoflavone 7-OMT has been cloned from alfalfa and the recombinant enzyme converts daidzein exclusively to isoformononetin when expressed in E. coli (He, et al. 1998. “Stress responses in alfalfa (Medicago sativa L.) XXII. cDNA cloning and characterization of an elicitor-inducible isoflavone 7-O-methyltransferase,” Plant Mol Biol 36: 43-54). This enzyme activity, and its corresponding transcripts, are strongly induced in elicited alfalfa cell cultures coordinately with other enzymes of medicarpin biosynthesis (Dalkin, et al. 1990. “Stress responses in alfalfa (Medicago sativa L.) I. Elicitor-induction of phenylpropanoid biosynthesis and hydrolytic enzymes in cell suspension cultures,” Plant Physiol 92: 440446; He, et al. 1998. Plant Mol Biol 36: 43-54).
A large number of plant OMT sequences are now available in the databases, most of which encode enzymes that act on hydroxycinnamic acid intermediates of lignin biosynthesis or on flavonoid derivatives (Ibrahim, et al. 1998. “Plant O-methyltransferases: molecular analysis, common signature and classification,” Plant Mol Biol 36: 1-10; Joshi, C. P. and V. L. Chiang. 1998. “Conserved sequence motifs in plant S-adenosyl-L-methionine-dependent methyltransferases,” Plant Mol Biol 37: 663-674). In nearly all cases, the enzymes exhibit strict regiospecificity; for example, a series of distinct, position-specific OMTs is involved in the synthesis of polymethylated flavonols in Chrysosplenium americanum (Ibrahim, et al. 1987. “Enzymology and compartmentation of polymethylated flavonol glucosides in Chrysosplenium americanum,” Phytochemistry 26: 1237-1245; Gauthier, et al. 1996. “cDNA cloning and characterization of a 3′/5′-O-methyltransferase for partially methylated flavonols from Chrysosplenium americanum,” Plant Mol Biol 32: 1163-1169). However, some OMTs appear to be more versatile, acting on both flavonoids and hydroxycinnamic acids (Gauthier, et al. 1998. “Characterization of two cDNA clones which encode O-methyltransferases for the methylation of both flavonoid and phenylpropanoid compounds,” Arch Biochem Biophys 351: 243-249), or are specific for more than one related substrate, such as the well-studied caffeic acid/5-hydroxyferulic acid OMTs of lignin biosynthesis (Bugos, et al. 1991. “cDNA cloning, sequence analysis and seasonal expression of lignin-bispecific caffeic acid/5-hydroxyferulic acid O-methyltransferase of aspen,” Plant Mol Biol 17: 1203-1215; Li, et al. 2000. “5-Hydroxyconiferyl aldehyde modulates enzymatic methylation for syringyl monolignol formation, a new view of monolignol biosynthesis in angiosperms,” J Biol Chem 275: 6537-6545). However, there have been no reports of OMTs, or any other plant natural product biosynthetic enzymes, that exhibit different regiospecificity in vivo and in vitro.
Utilizing the different regiospecificity of isoflavone 7-OMT in vivo, it is now possible to genetically manipulate biologically active 4′-O-methylated isoflavonoids.